Images

Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
  • C12N5/0629—Keratinocytes; Whole skin
  • C12N5/063—Kereatinocyte stem cells; Keratinocyte progenitors

Definitions

• This invention relates to a method of enriching for and isolating subpopulations of epithelia cells, isolation of keratinocyte stem cells, to keratinocyte stem cells and uses for keratinocyte stem cells.
• the human epidermis In common with other rapidly renewing tissues such as the haemopoietic system and the intestinal epithelia, the human epidermis is in a process of constant regeneration. Terminally differentiated cells lost continuously from the skin surface, are replaced by an intricate and highly regulated proliferative process within the basal layer of the epidermis. Stem cells in these rapidly renewing tissues are the earliest progenitors of a hierarchy of proliferative cells which are ultimately responsible for the generation of all mature cells for the lifetime of an individual (Lajtha, 1979).
• keratinocyte stem cells which represent a minor subpopulation of relatively quiescent cells, defined by their great proliferative potential and an unlimited capacity for self renewal, identified as slow-cycling, 3 H-Tdr label-retaining cells
• TA transit amplifying
• basal keratinocytes representing post-mitotic differentiating cells in the early stages of keralinisation can also be identified (Potten, 1983; Morris et al, 1985; MacKenzie & Bickenbach, 1985; Potten, 1986; Bickenbach et al, 1986; Christophers, 1971; Allen & Potten, 1974). Human epidermis has similar populations.
• multilineage reconstituting stem cells can be physically separated from committed progenitor cells (analogous to the TA cells of the epidermis). based upon differences in their expression of cell surface markers (Civin et at, 1984; Spangrude et al, 1988; Berenson et al, 1991; Terstappen et al, 1991; Baum et al, 1992). Clearly the availability of appropriate cell surface markers on basal epidermal cells would greatly facilitate the isolation and characterisation of human KSCs. However, the cell surface antigenic phenotype of these cells remains relatively poorly defined.
• Integrins are heterodimeric cell surface glycoproteins that primarily mediate the attachment of basal keratinocytes to extracellular matrix proteins found in the basement membrane, but can also mediate intercellular adhesion.
• basal keratinocytes express the ⁇ 1 integrins ⁇ 3 ⁇ 1 and as well as the integrin ⁇ 6 ⁇ 4 (Peltonen et at, 1989; Carter et al, 1990a Carter et al, 1990b).
• epidermal stem cells constitute between 1%-10% of the basal layer depending on the methodology used (Morris et al, 1985; MacKenzie & Bickenbach, 1985; Bickenbach et al, 1986; Potten & Hendry, 1973; Morris & Potten, 1994). Since approximately 40% of the basal layer in human foreskin exhibits high levels of ⁇ 1 integrin in vim (Jones et at, 1995) it is highly likely that basal keratinocytes with this phenotype contain both the KSC population and a significant number of TA cells and therefore there are drawbacks in the use of cells enriched for high level expression of ⁇ 1 .
• An object of one aspect of the invention is to generate a more purified population of keratinocyte stem cells than has been achieved by prior art methods.
• An object of a further aspect of the present invention is to provide methods for purifying subpopulations of epithelial cells.
• basal keratinocytes expressing low levels of ⁇ 6 ⁇ 4 represent a subpopulation of post-mitotic, differentiating keratinocytes
• this integrin is expressed at high levels on both the KSC and TA cells.
• this cell surface marker alone cannot be used to separate KSCs from TA cells to a high degree of purity but can do so to a degree of purity higher than where ⁇ 1 integrin is used.
• the invention could be said to reside in a method of enriching a viable population of KSCs from a population of epidermal cells comprising,
• TA cells might be purified from KSCs whereby a proportion of cells with low expression of a marker associated with proliferation are removed from the partially enriched pool.
• the epidermal cell population might be derived (torn a tissue sample of the skin. This method normally involves the separation of epidermis from the skin sample, before the enrichment.
• KSC cells One particularly good source of KSC cells is from the basal layer of the epidermis. The proportion of these cells that are KSCs will depend upon the type of skin, and the age of the individual concerned. It is estimated for example that about 10% of neonatal foreskins are KSC cells but a lesser proportion will be present in the basal layer of the epithelium of adults. Hair follicles are also known to be a reservoir of stem cells and might be used as a source rich in KSCs.
• ⁇ 1 is however less effective in the two step enrichment process than ⁇ 6 ⁇ 4 integrin because it recognises epidermal cells that have developed past the TA stage and therefore the first enrichment step leads to a lesser enrichment than by use of ⁇ 6 ⁇ 4 integrin which recognises only KSC cells and TA cells.
• the marker associated with proliferation that has been used by the inventors is one that is recognised by monoclonal antibody 1007 and has now been identified as being the transferrin receptor.
• monoclonal antibody 1007 There are a number of commercially available monoclonal antibody preparations that also recognise transferrin receptor.
• Alternative markets that are associated with proliferation can also be used examples of these include but are not limited to the EGF (Epidermal Growth Factor) receptor, and perhaps also the IGF (Insulin Growth Factor) receptor and the KGF (Keratinocyte Growth Factor) receptor.
• binding agent preferably being monoclonal antibodies or based on monoclonal antibodies because of the specificity of these latter agents.
• Antibodies can be used for both steps. However other agents might also be used, thus ligands for these integrins such as extracellular matrix proteins including laminin-5 or collagen I or IV may also be employed to enrich for cells carrying ⁇ 6 ⁇ 4 .
• transferrin itself could be used as a means for detecting the levels of transferrin receptor, in place of 10G7 or other antibody directed against transferrin receptor.
• the antibodies may be attached to a solid support to allow for crude separation.
• the separation techniques employed should maximise the retention of viability of the fraction to be collected.
• Various techniques of different efficacy may be employed to obtain relatively crude separations. The particular technique employed will depend upon efficiency of separation, associated cytoxicity, ease and speed of performance, and necessity for sophisticated equipment and/or technical skill.
• Procedures for separation may include, but are not limited to, magnetic separation, using antibody-coated magnetic beads, affinity chromatography and “panning” with antibody attached to a solid matrix.
• Techniques providing accurate separation include but are not limited to, FACS.
• ⁇ 6 ⁇ 4 has been selected on the basis of the portion being identified, i.e. ⁇ 6 , however the ⁇ 4 portion could equally well be used.
• Commercially available monoclonal antibody preparations that recognise ⁇ 6 are available for these for example those known as GOH3 and 4F10.
• a purified KSC cell population can be achieved, which is believed to have less than 1% non KSC cells, however using cruder enrichment techniques a variety of levels of purified KSC cells can be produced, and may be useful at lower levels of purity. It is believed that purity levels of greater than 50% or at least greater than 70% may be useful for an improved formation of an epidermal layer which can be used as a graft for a skin lesion.
• a substantially purified KSC cell population of greater than about 90% is thought to be useful for not only the formation of an epidermal layer but also for use as a starting cell population for genetic modification whereby exogenous nucleic acid is introduced to express a desired product, which may be used in gene therapy.
• KSCs an ideal candidate for generic manipulation and gene therapy for the treatment of both skin disorders and systemic deficiencies.
• exogenous nucleic acid would be introduced into autologous KSCs, to produce a therapeutically useful substance.
• the cells can be formed into an epidermal layer which could be grafted onto the skin of the individual concerned and act as a means for long term release of the therapeutic compound.
• the compound is thus introduced systemically.
• the compound might be one that is not produced by the individual as a result of a congenic defect, or a disorder that has developed, for example diabetes.
• Purification of KSCs according to the present invention are suggested to give rise to a skin graft of greater persistence and accordingly the gene therapy should last longer than would otherwise be possible. Suggestions for gene therapy using KSCs for gene therapy have been made with one of the acknowledged deficiencies being recognised as the lack of purified KSCs.
• a third useful result of this invention relates to the enhanced capacity to find markers associated with proliferation of various cell sub populations of the skin involved in the early proliferative events and to enhance the understanding of these early proliferative events, with the greater potential to discover the reason for defects in the proliferative process which lead to cancers.
• compositions including an enriched cell population of KSC cells capable of being enriched by firstly enriching a cell population for cells that carry an integrin marker and secondly reducing the number of cells that carry a marker associated with proliferation.
• compositions might include a tissue layer suitable for autologous skin graft application and a genetically modified population of KSC cells.
• the invention could be said to reside in a composition formed from an enriched cell population of KSC of this invention.
• the composition could be the result of an epidermal layer that is used as a skin graft that has been derived from KSCs.
• FIGS. 1 A and 1 B Fractionation and colony forming ability of neonatal primary human foreskin basal epidermal cells on the basis of ⁇ 6 integrin expression.
• FIG. 1 A Flow cytometric analysis of freshly isolated basal keratinocytes stained with either an anti- ⁇ 6 Mab (4F10 solid line) or isotype control Mab (ID4.5-broken line), detected by a FITC-conjugated secondary antibody. Two fractions representing the upper 30% (R 6 ) ⁇ 6 bri cells and the lower 30% (R 7 ) ⁇ 6 dim cells were collected by FACS and cultured.
• FIGS. 2 A and 2 B Long-term growth capacity of ⁇ 6 bri and ⁇ 6 dim cells.
• FIG. 2 B The total cell output (cumulative cell yield), from 5000 cells of each fraction, was determined at the end of the experiment when their ability to proliferate was exhausted.
• the total cell output of the ⁇ 6 bri fraction was significantly higher than the ⁇ 6 dim fraction and UF cells (p ⁇ 0.05), thus confirming that the ⁇ 6 bri population had the greatest long term proliferative capacity.
• the numbers above the columns indicate mean cell yields from each fraction. The results shown represent the mean total output ⁇ SEM of three separate experiments.
• FIGS. 3 A and 3 B Two-colour flow cytometric analysis of ⁇ 6 and keratins 14 and 10 in neonatal primary human foreskin basal epidermal cells.
• Freshly isolated keratinocytes were fixed, permeabilised and double labeled with anti- ⁇ 6 integrin (Mate 4F10) and either (A) anti-K14 (Mate LL001 or (B) anti-K10 (Mate LHP2).
• Cells were analysed for keratin expression after gating into ⁇ 6 bri and ⁇ 6 dim fractions.
• FIG. 3 A shows that both these fractions were positive for the basal keratin K14, but that the ⁇ 6 dim cells expressed lower levels of K14 than the ⁇ 6 bri cells.
• FIG. 3 B shows that the ⁇ 6 bri fraction was negative for the differentiation-specific keratin K10, while ⁇ 6 dim cells were positive for this marker. Staining with appropriate isotype-matched negative control Mabs (ID4.5 and IB5) is shown (dotted lines) in each figure.
• FIGS. 4 A and 4 B Fractionation and colony forming ability of neonatal primary human foreskin basal epidermal cells on the basis of ⁇ 6 integrin and 10G7 ag expression.
• FIG. 4 A Dot plot showing flow cytometric analysis of freshly isolated basal keratinocytes double-labeled with anti- ⁇ 6 Mab 4F10 (FITC), and Mab 10G7 (PE), from a representative experiment.
• FITC anti- ⁇ 6 Mab 4F10
• PE Mab 10G7
• FIG. 4 B Colony numbers obtained from 5000 cells from each fraction at two weeks in culture.
• the ⁇ 6 bri 10G7 dim and ⁇ 6 bri 10G7 bri fractions consistently gave rise to greater colony numbers than the ⁇ 6 dim fraction.
• Importantly no significant difference between colony numbers was obtained from the ⁇ 6 bri 10G7 dim and ⁇ 6 10G7 bri fractions.
• FIGS. 5A and 5B Long-term growth capacity of primary human neonatal foreskin basal epidermal cells fractionated on the basis of ⁇ 6 and 10G7 ag expression.
• FIG. 5 A Growth curves of UF, ⁇ 6 bri 10G7 dim and ⁇ 6 bri 10G7 bri fractions in a representative experiment. The curves show that the ⁇ 6 bri 10G7 dim cells consistently grew at a greater rate than the UF and ⁇ 6 bri 10G7 bri cells. Cell output at earlier time points (Day 0 - 50 ) is shown in the inset, and indicates that all fractions were capable of growth in culture, not evident on the main graph due to the scale. Data points represent mean ⁇ SEM of three replicates.
• FIG. 5 B Total cell output of fractions determined at the end of the experiment, from an initial input of 5000 cells per fraction, confirm that the ⁇ 6 bri 10G7 dim population has the greatest long-term proliferative capacity and comprises the candidate keratinocyte stem cell population.
• FIG. 6 Cell cycle analysis of primary basal keratinocytes fractionated on the basis of ⁇ 6 and 10G7 ag expression.
• the UF cells show that overall, the basal layer contains about 5% of cells progressing through the S-phase of the cell cycle.
• Analysis of fractionated cells clearly demonstrates that the majority of these actively cycling basal cells reside within the candidate TA population ( ⁇ 6 bri 10G bri cells), whereas the candidate KSC population ( ⁇ 6 bri 10G7 dim ) and the post-mitotic differentiating (PM-D) cells ( ⁇ 6 dim fractions) comprise mostly quiescent cells, with relatively fewer cells in S- or S/G 2 M phase.
• the results displayed in this figure are the mean ⁇ SEM of four separate experiments.
• FIG. 7 Total cell output of ⁇ 6 bri and ⁇ 1 bri further subdivided with Mab 10G7. The values above each bar represent mean cell yield from each fraction.
• FIG. 8 Is a graphical representation of telomerase activity in fractionated human keratinocytes, as measured by absorbance at 450 nm using an ELISA assay (Kim et al, 1994).
• PM-D refers to Post-mitotic differentiating cells
• TA refers to transit amplifying cells
• KSCs refers to keratinocyte stem cells
• UF refers to unfractionated cells.
• FIG. 9 Is a similar plot to that shown in FIG. 4 except that the cells harvested were from the facial skin of a 72 year old adult. What is shown is a dot plot showing flow cytometric analysis of freshly isolated basal keratinocytes double-labelled with anti- ⁇ 6 Mab 4F10 (FITC), and Mab 10G7 (PE), from a representative experiment.
• PM-D refers to Post-mitotic differentiating cells ( ⁇ 6 dim )
• TA refers to transit amplifying cells ( ⁇ 6 bri 10G7 bri )
• KSCs refers to keratinocyte stem cells( ⁇ 6 bri 10G7 dim ) This phenotype has been observed in numerous replicate experiments. The populations of cells which are similar to those observed in neonatal epidermis are indicated in the boxes.
• Keratinocytes were cultured using the Rheinwald and Green method (Rheinwald & Green, 1975), on irradiated Swiss 3T3-J2 feeder layers in DMEM containing 10% FCS, 20 ng/ml epidermal growth factor (Sigma), 0.4 ⁇ g/ml hydrocortisone (Sigma), and 10 ng/ml cholera toxin (Calbiochem La Jolla, USA). Keratinocytes were passaged after removing the feeder cells with 0.02% EDTA.
• Mab 4F10 (IgG 2a ) to the ⁇ 6 integrin subunit was used at 20 ⁇ g/ml (Serotec, Oxford, UK); Mab 10G7 (IgG 2a ) developed in our laboratory was used as undiluted hybridoma supernatant.
• And-mouse IgG 2b -FITC and IgG 2a -PE were used to detect 4F10 and 10G7 binding respectively.
• Mabs LHP2 (IgG 1 ) to K10 and LL001 (IgG 2a ) to K14 were kindly provided by Dr Irene Leigh (Royal London Hospital, London, UK), and used at 1:10 and 1:1000 respectively.
• Basal keratinocytes were processed for single ( ⁇ 6 -FITC) or double ( ⁇ 6 -FITC and 10G7 ag)-PE staining along with appropriate negative controls and single colour positive controls to establish compensation settings on the FACS as described previously. (Kaur et al, 1997).
• the cells were resuspended in culture medium at 2-3 ⁇ 10 6 /ml, sorted using the Becton-Dickinson FACStar Plus and collected into culture medium. The viability of the cells after sorting was determined to be >95%. Double staining for keratins and ⁇ 6 was performed on cells fixed and permeabilised in 70% ethanol at ⁇ 20° C. for 10 mins.
• the KSC population has been defined as a minor subpopulation of the basal layer with greatest proliferative capacity since it must sustain tissue renewal for a lifetime. Based on the assumption that KSCs have a specific cell surface phenotype, as has been demonstrated for bone marrow haemopoietic progenitors, when plating equivalent numbers of cells with the hypothesised KSC phenotype and UF cells, one should clearly expect greater cell output from the former population since it has been enriched for stem cells. At the start of each long term culture experiment, cells fractionated on the basis of their cell surface phenotype were sorted. 5000 keratinocytes from each fraction were plated into 24-well plates containing monolayers of feeder cells.
• Fractionated primary basal keratinocytes were collected by FACS, fixed with 70% ethanol (20° C.), and treated with RNAase prior to staining with 40 ⁇ g/ml propidium iodide. DNA content was analysed by flow cytometry on an EPICS XL flow cytometer (Couker) within 12 hours.
• stem cells may not maintain their in vivo characteristics in culture after removal from their “niche” or microenvironment (Schofield et al, 1978), we elected to analyse freshly isolated primary epidermal cells.
• the KSC subpopulation defined by its relative quiescence in viva and the greatest proliferative potential in vitro; compared to the TA cells characterized by their actively cycling status in vivo, reduced proliferative potential and more rapid terminal differentiation in culture (Lajtha, 1979).
• K keratins
• K 5 and K 14 are expressed by basal cells
• K 1 and K 10 are predominantly expressed in the suprabasal differentiating layers of the epidermis (Fuchs & Green. 1980).
• K 10 expression has also been observed in a minor subpopulation of basal cells in murine epidermis (Schweizer, 1984; Mackenzie et at, 1989) suggesting the presence of differentiating cells within the basal layer.
• the ⁇ 6 dim fraction In contrasts while the ⁇ 6 bri keratinocytes were negative for K 10 , the ⁇ 6 dim fraction expressed this differentiation marker (FIG. 3 B).
• the ⁇ 6 dim fraction comprise a population of post-mitotic differentiating basal cells, while the ⁇ 6 bri fraction contains the majority of proliferative basal keratinocytes i.e. KSCs and TA cells.
• Human epidermal stem cells can be resolved further within the ⁇ 6 bri population on the basis of 10G7 antigen (transferrin) expression.
• total cell outputs from 5000 UF, ⁇ 6 bri , ⁇ 6 bri 10G7 bri or ⁇ 6 bri 10G7 dim were 7 ⁇ 10 5 , 1.9 ⁇ 10 7 , 1.4 ⁇ 10 8 and 5.5 ⁇ 10 8 respectively.
• the absolute number of cells generated by a particular fraction in long-term culture was variable between experiments and can be attributed to variation between skin donors, and the duration of the experiment, dictated by the period for which cells from a particular donor could be maintained in culture.
• the ⁇ 6 bri 10G7 dim fraction consistently contained basal epidermal cells with the greatest total cell output in several replicate experiments (n ⁇ 5) (see Table 1).
• the candidate keratinocytes stem cell fraction ( ⁇ 6 bri 10G7 dim ) represents a quiescent subpopulation of the epidermal basal layer.
• Basal keratinocytes with the phenotype ⁇ 6 10G7 dim have important stem cell attributes.
• the present strategy for enriching for epidermal stem cells on the basis of a proliferation-related cell surface marker allows for the separation of proliferative basal cells into the quiescent KSC compartment demonstrating the greatest regenerative capacity in long-term culture ( ⁇ 6 bri 10G7 dim ) and an actively cycling TA compartment with lesser proliferative capacity ( ⁇ 6 bri 10G7 bri ).
• ⁇ 6 bri 10G7 dim a single candidate KSC with the phenotype ⁇ 6 bri 10G7 dim can generate approximately 5.8 ⁇ 10 8 cells. It is highly likely that the present culture conditions, while promoting very effective growth of the TA population, do not permit optimal cell generation from or self-renewal of the KSC population.
• the present data also show that the candidate KSC fraction represents an immature and minor subpopulation of approximately 10% of the basal layer, consistent with estimates of 1-10% from kinetic studies in murine epidermis (Morris et al, 1985; MacKenzie & Bickenbach, 1985; Potten, 1986: Bickenbach et al, 1986; Potten & Hendry 1973). Given that we have used neonatal human foreskin tissue which is capable of greater proliferation than adult foreskin epithelium (Rheinwald & Green, 1975), it is likely that these KSC numbers are higher than may be found in adult epidermis.
• telomere end nucleotide repeat sequences which are normally lost from the ends of chromosomes with replication.
• Telomerase activity has been round to be present in a great number of actively proliferating cells including tumour and normal cell lines in culture, and its is now well accepted that there is a good correlation between proliferative cells and telomerase activity (Greider, 1998). It was originally proposed that stem cells which are long lived may contain high levels of telomerase enzyme although it has become evident that haemopoietic committed progenitors (the actively proliferating cells) have relatively high levels of this enzyme, while minimal to undetectable levels are present within the stern cells (Hiyama et al. 1995).
• KSCs as defined by us as cells with the phenotype ⁇ 6 bri 10G7 dim express significantly lower levels of telomerase compared to the actively proliferating TA population (phenotype ⁇ 6 10g7 bri ) as shown in FIG. 8 .
• These experiments utilized a TRAP assay telomerase activity as described by Kim et al, (1994) detected by ELISA. The interpretation we favour is that telomerase activity is not essential in normally quiescent stem cells, but is activated during cell cycling.
• the ability to regenerate epithelium in vivo is an important property of KSCs, and an important prerequisite to the development of gene therapeutic approaches aiming to deliver gene products from skin grafts.
• This proposed example includes the steps of taking purified KSCs to generate epidermal tissue, which can then be utilised to graft onto individuals.
• Cultures can be processed for sectioning and immunohistochemical analysis for various epithelial markets including integrins, keratins, involucrin, filaggrin etc.
• Organotypic cultures will be generated from fractionated basal epidermal cells, (together with unfractionated cells as controls), from both adult and neonatal skin. It is postulated that only the ⁇ 6 bri 10G7 dim fraction will be capable of generating a normal epithelium with a basal layer and differentiated layers, and that the ⁇ 6 dim (and perhaps ⁇ 6 bri 10G7 bri ) fraction will give rise to terminally differentiated cells.

Landscapes

• Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Life Sciences & Earth Sciences (AREA)
• Biomedical Technology (AREA)
• Wood Science & Technology (AREA)
• Organic Chemistry (AREA)
• Chemical & Material Sciences (AREA)
• Biotechnology (AREA)
• Zoology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Genetics & Genomics (AREA)
• Microbiology (AREA)
• Cell Biology (AREA)
• Dermatology (AREA)
• Biochemistry (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Developmental Biology & Embryology (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Applications Claiming Priority (3)

Related Parent Applications (1)

Publications (1)

Family

ID=3806706

Family Applications (1)

Country Status (4)

Cited By (7)

Families Citing this family (5)

Citations (1)

• 1998
  • 1998-03-18 AU AUPP2444A patent/AUPP244498A0/en not_active Abandoned
• 1999
  • 1999-03-18 GB GB0022845A patent/GB2351296B/en not_active Expired - Fee Related
  • 1999-03-18 WO PCT/AU1999/000177 patent/WO1999047644A1/fr active IP Right Grant
• 2000
  • 2000-09-18 US US09/664,437 patent/US6485971B1/en not_active Expired - Lifetime

Patent Citations (1)

Non-Patent Citations (48)

Also Published As

Similar Documents

Legal Events